The GO system includes 7,8-dihydro-8-oxoguanine, the structure of the predominant tautomeric form of the GO lesion. Oxidative damage can lead to GO lesions in DNA. MutY removes the misincorporated adenine from the A/GO mispairs that result from error-prone replication past the GO lesion. Repair polymerases are much less error-prone during trans lesion synthesis and can lead to a C/GO pair. Oxidative damage can also lead to 8-oxo-dGTP. Inaccurate replication could result in the misincorporation of 8-oxo-dGTP opposite template A residues, leading to A/GO mispairs. MutY could be involved in the mutation process because it is active on the A/GO substrate and would remove the template A, leading to the AT.RTM. CG transversions that are characteristic of a MutT strain. The 8-oxo-dGTP could also be incorporated opposite template cytosines, resulting in a damaged C/GO pair that could be corrected by MutM.
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention has been putatively identified as a human homologue of the E. coli MutY gene, sometimes hereinafter referred to as "hMYH".
Mismatches arise in DNA through DNA replication errors, through DNA recombination, and following exposure of DNA to deaminating or oxidating environments. Cells have a host of strategies that counter the threat to their genetic integrity from mismatched and chemically damaged base pairs (Friedberg, E C, DNA repair, W. H. Freeman, New York (1985)). With regard specifically to mismatch repair of replication errors, Escherichia coli and Salmonella typhimurium direct the repair to the unmethylated newly synthesized DNA strand by dam methylation at d(GATC) sequences, using the MutHLS systems (Clavery, J. P. and Lacks, S. A., Microbiol., Rev. 50:133-165 (1986); Modrich, P. Annu. Rev. Genet. 25:229-253 (1991); Radman, M. and R. Wagner, Annu. Rev. Genet. 20:523-528 (1986)). The very short patch pathway of E. coli is specific for a correction of T/G mismatches (a mismatch indicated by a slash) and is responsible for the correction of deaminated 5-methylcytosine (Jones, M., et al., Genetics, 115:605-610 (1987); Lieb, M., Mod. Gen. Genet. 181:118-125 (1983); Lieb, M., and D. Read, Genetics 114:1041-1060 (1986); Raposa, S. and N. S. Fox, Genetics 117:381-390 (1987)).
The E. coli MutY pathway corrects A/G and A/C mismatches, as well as adenines paired with 7,8-dihydro-8-oxo-deoxyguanine (8-oxoG or GO) (Au, K. G., et al., Proc. Natl. Acad. Sci. U.S.A. 85:9163-9166 (1988); Lu, A. L. and D. Y. Chang, Genetics, 118:593-600 (1988); Michaels, M. L., et al., Proc. Natl. Acad. Sci. U.S.A., 89:7022-7025 (1992); Michaels, M. L., et al., Biochemistry, 31:10964-10968 (1992); Radicella, J. P., et al., Proc. Natl. Acad. Sci. U.S.A., 85:9674-9678 (1988); Su, S. -S., et al., J. Biol. Chem. 263:6829-6835 1988). The 39-kDa MutY protein shares some homology with E. coli endonuclease III and contains a [4Fe-4S].sup.2+ cluster (Lu, A. -L., et al., 1994, Unpublished data; Michaels, M. L., et al., Nucleic Acids Res. 18:3841-3845 (1990); Tsai-Wu, J. -J., et al., Proc, Natl. Acad. Sci. U.S.A. 89:8779-8783 (1992); Tsai-Wu, J. -J., et al., J. Bacteriol. 173:1902-1910 (1991)). The MutY preparation of Tsai-Wu et al. (Tsai-Wu, J. -J., et al., Proc. Natl. Acad. Sci. U.S.A. 89:8779-8783 (1992)) has both DNA N-glycosylase and apurinic or apyrimidinic (AP) endonuclease activities, whereas those purified by Au et al. (Au K. G., et al., Proc. Natl. Acad. Sci. U.S.A., 86:8877-8881 (1989), and Michaels et al. (Michaels, M. L., et al., Proc. Natl. Acad. Sci. U.S.A., 89:7022-7025 (1992); Michaels, M. L., et al., Biochemistry, 31:10964-10968 (1992) possess only the glycosylase activity. DNA glycosylase specifically excises the mispaired adenine from the mismatch and the AP endonuclease cleaves the first phosphodiester bond 3' to the resultant AP site (Au K. G., et al., Proc. Natl. Acad. Sci. U.S.A., 86:8877-8881 (1989); Tsai-Wu, J. -J., et al., Proc, Natl. Acad. Sci. U.S.A. 89:8779-8783 (1992).
Repair by the MutY pathway involves a short repair tract and DNA polymerase I (Radicella, J. P., et al., J. Bacteriol., 175:7732-7736 (1993); Tsai-Wu, J. -J., and A. -L. Lu, Mol. Gen. Genet. 244:444-450 (1994)).
The mismatch repair strategy detailed above has been evolutionarily conserved. Genetic analysis suggests that Saccharomyces cerevisiae has a repair system analogous to the bacterial dam methylation-dependent pathway (Bishop, D. K., et al., Nature (London) 243:362-364 (1987); Reenan, R. A. and R. D. Kolodner, Genetics, 132:963-973 (1992); Reenan, R. A. and R. D. Kolodner, Genetics, 132:975-985 (1992); Williamson, M., et al., Genetics, 110:609-646 (1985)). This pathway is functionally homologous to the E. coli very sort patch pathway for the correction of deaminated 5-methylcytosine.
Two mutator genes in E. coli, the mutY and the mutM genes (Cabrera et al., J. Bacteriol., 170:5405-5407 (1988); and Nghiem, Y., et al., PNAS, U.S.A., 85:9163-9166 (1988)) have been described, which work together to prevent mutations from certain types of oxidative damage, dealing in particular with the oxidized guanine lesion, 8-oxodGuanine (Michaels et al., PNAS, U.S.A., 89:7022-7025 (1992). In Michaels, M. L., and Miller, J. H., J. Bacteriol., 174:6321-6325 (1992) is a summary of the concerted action of these two enzymes, both of which are glycosylases. The MutM protein removes 8-oxodG from the DNA, and the resulting AP site is repaired to restore the G:C base pair. Some lesions are not repaired before replication, which results in 50% insertion of an A across from the 8-oxodG, which can lead to a G:C to T:A transversion at the next round of replication. However, the MutY protein removes the A across from 8-oxodG and repair synthesis restore a C most of the time, allowing the MutM protein another opportunity to repair the lesion. In accordance with this, mutators lacking either the MutM or MutY protein have an increase specifically in the G:C to T:A transversion (Cabrera et al., Id., (1988); and Nghiem, Y., et al., Id. (1988)), and cells lacking both enzymes have an enormous increase in this base substitution (Michaels et al., Id. (1992). A third protein, the product of the mutT gene, prevents the incorporation of 8-oxodGTP by hydrolyzing the oxidized triphosphate back to the monophosphate (Maki, H., and Sekiguchi, M., Nature, 355:273-275 (1992)), preventing A:T to C:G transversions.
Accordingly, there exists a need in the art for identification and characterization of genes and proteins which modulate the human cellular mutation rate, for use as, among other things, markers in cancer and diseases associated with DNA repair. In particular, there is a need for isolating and characterizing human mismatch repair genes and proteins, which are essential to proper development and health of tissues and organs, such as the colon, and which can, among other things, play a role in preventing, ameliorating, diagnosing or correcting dysfunctions or disease, particularly cancer, and most particularly colon cancer, such as, for example, HNPCC (non-polyposis colon cancer).
In accordance with one aspect of the present invention, there is provided a novel mature polypeptide, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptide of the present invention is of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding a polypeptide of the present invention including mRNAs, cDNAs, genomic DNAs as well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of the present invention, under conditions promoting expression of said protein and subsequent recovery of said protein.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide, for therapeutic purposes, for example, to repair oxidative damage to DNA and prevent mutations from oxidative lesions, treat genetic diseases related to a mutated hMYH gene, for example, xerederma pigmentosum and neoplasia, and to diagnose an abnormal transformation of cells, particularly cancer, and most particularly colon cancer, such as for example HNPCC, and/or to diagnose a susceptibility to abnormal transformation of cells, particularly cancer, and most particularly colon cancer, such as for example HNPCC.
In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.
In accordance with yet a further aspect of the present invention, there is also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases or susceptibility to diseases related to mutations in the nucleic acid sequences encoding a polypeptide of the present invention. In accordance with a further aspect of the invention is a process for diagnosing a cancer comprising determining from a sample derived from a patient a decreased level of activity of polypeptide having the sequence of SEQ ID NO: 2.
In accordance with a further aspect of the invention is a process for diagnosing a cancer comprising determining from a sample derived from a patient a decreased level of expression of a gene encoding a polypeptide having the sequence of SEQ ID NO: 2.
In accordance with a further aspect of the invention is a process for diagnosing a cancer comprising determining from a sample derived from a patient a decreased level of activity of polypeptide having the sequence of SEQ ID NO: 9.
In accordance with a further aspect of the invention is a process for diagnosing a cancer comprising determining from a sample derived from a patient a decreased level of expression of a gene encoding a polynucleotide having the sequence of SEQ ID NO:9.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, for example, synthesis of DNA and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.